Feeding Strategies to Mitigate Enteric Methane Emission from Ruminants in Grassland Systems

Simple Summary Ruminants under grazing conditions play an important role, especially in developing countries. Enteric methane emissions from ruminants are greater with pasture-based diets; however, it is not clear which abatement practices are effective to reduce methane emissions under grazing conditions. The objective of this review was to identify and describe enteric methane abatement practices for ruminants that are applicable under grazing conditions. Decreasing the pre-grazing herbage mass reduced methane emissions per unit of product. Other grazing management practices such as increased stocking rate, decreased forage maturity, rotational stocking, and incorporating tannin-containing or non-tannin-containing legumes showed inconsistent results. Nitrogen fertilization or silvopastoral systems did not modify methane emissions, although they may alter carbon sequestration in a system. Supplementation in grazing conditions shows inconsistent responses on methane emissions. However, lipid supplementation showed promising results. Identifying and implementing grazing strategies and supplementation practices under grazing conditions is required to increase efficiency and reduce the environmental impact of these systems. Abstract Ruminants produce approximately 30% of total anthropogenic methane emissions globally. The objective of this manuscript was to review nutritional enteric methane abatement practices for ruminants that are applicable under grazing conditions. A total of 1548 peer-reviewed research articles related to the abatement of enteric methane emissions were retrieved and classified into four categories: non-experimental, in vitro, in vivo confined, and in vivo grazing. The methane abatement strategies for grazing systems were arranged into grazing management and supplementation practices. Only 9% of the retrieved papers have been conducted under grazing conditions. Eight grazing management practices have been evaluated to reduce methane emissions. Decreasing the pre-grazing herbage mass reduced the methane emission per unit of product. Other grazing management practices such as increased stocking rate, decreased forage maturity, rotational stocking, and incorporating tannin-containing or non-tannin-containing feeds showed contradictory results. Nitrogen fertilization or silvopastoral systems did not modify methane emissions. Conversely, supplementation practices in grazing conditions showed contradictory responses on methane emissions. Lipid supplementation showed promising results and suggests applicability under grazing conditions. Identifying and implementing grazing strategies and supplementation practices under grazing conditions is required to increase efficiency and reduce the environmental impact of these systems.

tation [14,25,26]. This manuscript aims to critically review nutritional abatement strategies of enteric CH 4 from ruminants in grassland systems reported in the scientific literature.
Non-experimental: Represented by documents that did not involve original research data collection, for example, reviews, meta-analyses, life cycle assessments, inventory estimations, or methodology description. ii. In vitro: This category comprised research that evaluated CH 4

emissions in batch or
semi-continuous in vitro cultures. iii. Confined studies: Represented by documents that evaluated in vivo CH 4

emissions,
where ruminants were restricted to confined facilities. iv. Grazing studies: This category includes research that determined in vivo CH 4 emissions under grazing conditions.
Retrieved documents were categorized according to the continent where the experiments were conducted (i.e., the Americas, Africa, Asia, Europe, or Oceania), production system (i.e., beef, dairy, or small ruminants), type of forage (i.e., temperate or tropical forage), and CH 4 measurement technique.
Research evaluating CH 4 emissions from ruminants published in the last decade increased by 6 and 28-fold relative to the decades 2001-2010 and 1990-2000, respectively ( Figure 1). Thus, there has been an increase in interest in measuring and evaluating strategies to reduce CH 4 emissions from ruminants in the last decade. Beauchemin et al. [25] reported similar tendencies, emphasizing that early CH 4 research focused more on increasing animal energy efficiency, understanding methanogenesis biochemical pathways, and evaluating rumen modifiers under in vitro or confined conditions. After the development of the sulfur hexafluoride (SF 6 ) tracer technique [12], and more recently, the use of the GreenFeed (GF, C-Lock Inc., Rapid City, SD) technology [27], more experiments have been conducted under grazing conditions [25]. mentation [14,25,26]. This manuscript aims to critically review nutritional abatement strategies of enteric CH4 from ruminants in grassland systems reported in the scientific literature.
Non-experimental: Represented by documents that did not involve original research data collection, for example, reviews, meta-analyses, life cycle assessments, inventory estimations, or methodology description. ii.
In vitro: This category comprised research that evaluated CH4 emissions in batch or semi-continuous in vitro cultures. iii.
Confined studies: Represented by documents that evaluated in vivo CH4 emissions, where ruminants were restricted to confined facilities. iv.
Grazing studies: This category includes research that determined in vivo CH4 emissions under grazing conditions. Retrieved documents were categorized according to the continent where the experiments were conducted (i.e., the Americas, Africa, Asia, Europe, or Oceania), production system (i.e., beef, dairy, or small ruminants), type of forage (i.e., temperate or tropical forage), and CH4 measurement technique.
Research evaluating CH4 emissions from ruminants published in the last decade increased by 6 and 28-fold relative to the decades 2001-2010 and 1990-2000, respectively ( Figure 1). Thus, there has been an increase in interest in measuring and evaluating strategies to reduce CH4 emissions from ruminants in the last decade. Beauchemin et al. [25] reported similar tendencies, emphasizing that early CH4 research focused more on increasing animal energy efficiency, understanding methanogenesis biochemical pathways, and evaluating rumen modifiers under in vitro or confined conditions. After the development of the sulfur hexafluoride (SF6) tracer technique [12], and more recently, the use of the GreenFeed (GF, C-Lock Inc., Rapid City, SD) technology [27], more experiments have been conducted under grazing conditions [25].  1951-1960 1961-1970 1971-1980 1981-1990 1991-2000 2001-2010 2011-2020 Publication number Year Non-experimental In Vitro Confined Grazing Publications involving reviews, meta-analyses, methodology description, and theoretical analyses (i.e., non-experimental category) represented 23% of the retrieved documents. In vitro publications were 31% of the documents, with the batch culture methodology being predominant (reported in 83% of the in vitro studies). The in vivo Confined category represented 39% of the documents, and respiration chambers, SF 6 , and GF techniques used in confinement studies were 57, 20, and 7% of the documents, respectively. Further, 16% of the confined studies provided cut and carry forage as a proxy for grazing conditions. The in vivo Grazing category represented 7% of the documents (Figure 2), and the SF 6 technique was used in 70% of the grazing research on CH 4 abatement. Research on enteric CH 4 emissions under grazing conditions has been carried out mainly in Europe and the Americas (67%), evaluating mostly cattle (83%) under temperate pastures (76%) (Figure 3). Publications involving reviews, meta-analyses, methodology description, and theoretical analyses (i.e., non-experimental category) represented 23% of the retrieved documents. In vitro publications were 31% of the documents, with the batch culture methodology being predominant (reported in 83% of the in vitro studies). The in vivo Confined category represented 39% of the documents, and respiration chambers, SF6, and GF techniques used in confinement studies were 57, 20, and 7% of the documents, respectively. Further, 16% of the confined studies provided cut and carry forage as a proxy for grazing conditions.
The in vivo Grazing category represented 7% of the documents (Figure 2), and the SF6 technique was used in 70% of the grazing research on CH4 abatement. Research on enteric CH4 emissions under grazing conditions has been carried out mainly in Europe and the Americas (67%), evaluating mostly cattle (83%) under temperate pastures (76%) ( Figure 3).

Methane Emissions in Grassland Systems
Grassland systems vary from extensive, generally with low productivity per area, low forage nutritive value, non-improved ruminant breeds, and without supplementation schemes, to intensive, generally with high productivity, improved animal breeds, grasses, and management, and more balanced diets [7,26]. This diversity in production systems requires the development of different approaches to reduce enteric CH4 emissions that will have a minimal effect on farm labor activities, production costs, and profitability. For example, continuous inclusion of a feed additive may be easily incorporated in intensive 17

Methane Emissions in Grassland Systems
Grassland systems vary from extensive, generally with low productivity per area, low forage nutritive value, non-improved ruminant breeds, and without supplementation schemes, to intensive, generally with high productivity, improved animal breeds, grasses, and management, and more balanced diets [7,26]. This diversity in production systems requires the development of different approaches to reduce enteric CH 4 emissions that will have a minimal effect on farm labor activities, production costs, and profitability. For example, continuous inclusion of a feed additive may be easily incorporated in intensive grazing systems through supplemental feed (e.g., during the milking routine). However, in extensive grazing conditions, daily additive supplementation is less feasible, and other strategies must be developed to ensure continuous CH 4 abatement.
From a feed and nutritional perspective, strategies to decrease CH 4 emissions can be classified into practices related to grazing management or strategic supplementation of ingredients or additives. Managing grazing intensity-targeted through contrasting stocking rates or pre-grazing herbage masses-and concentrate supplementation have been the most reported strategies to modulate CH 4 under grazing conditions (12 and 9 studies, respectively). This was followed by lipid (6 studies) and nitrate supplementation (3 studies). Other strategies that modify forage nutritive value (i.e., incorporation of tannin-containing legumes or manipulation of forage maturity) have been less evaluated (2 studies each).

Grazing Management Strategies to Mitigate Methane Emissions from Ruminants
Grazing management consists of practices that manipulate forage characteristics to pursue one or multiple objectives. Grazing intensity, phenological stage of grasses, and grazing method are management tools that manipulate forage availability or quality [28]. Grazing intensity, generally expressed as stocking rate, or relationship between the number of animals and amount of land grazed, is the main factor affecting nutrient cycling, and defines animal productivity while delivering ecosystem services [28,29]. Under grazing conditions, increasing the stocking rate showed inconsistent results on CH 4 emissions (Table 1). However, methane intensity was not affected by increasing the stocking rate (Table 1). Increasing the stocking rate decreases forage biomass, but increases forage nutritive value [59], as greater stocking rates are associated with extensive biomass defoliation, maintaining the vegetative stage of forages [28]. Consumption of forage material in a vegetative stage is associated with reduced CH 4 yield due to a lesser concentration of cell wall structure [43]. Additionally, intake of forages in a vegetative stage increases dry matter consumption, decreases ruminal retention time, and reduces CH 4 yield (i.e., grams of CH 4 per unit of dry matter intake), although it could increase total CH 4 production (i.e., total grams of CH 4 per animal per day [60]). Conversely, individual animal productivity decreases when the stocking rate increases due to greater grazing competition and less possibility for animal forage selection [61]. The intensity or yield of enteric CH 4 emissions may be increased with high stocking rates if forage availability is restricted and is insufficient to meet the animal's nutrient requirements.
The phenological stage of grazing is related to the physiological stage of forage, i.e., forage maturity, when the defoliation occurs [28]. Reduced forage maturity decreased the CH 4 conversion factor (i.e., Ym; Table 1). Mature forages have a lesser concentration of digestible tissues such as parenchyma and more cell wall structure. This structure is mainly composed of cellulose, hemicellulose, and lignin and has slow rumen fermentation and passage rates [62]. The fermentation of cell wall carbohydrates yields more CH 4 than non-structural carbohydrates [63]. Conversely, immature forages have more degradable nutrients and result in increased dry matter intake [42]. Thus, it is expected that mature forages in ruminant diets produce a greater CH 4 yield due to greater cell wall concentration. However, if the forage cell wall structure limits intake (i.e., physical restriction), the CH 4 production may be reduced, increasing CH 4 yield [60].
The grazing method refers to how animals are stocked and is generally classified as continuous or rotational [64]. Usually, rotational grazing is associated with more uniformly distributed grazing and manure deposition, increasing the carrying capacity and efficiency of grazing, maintaining forage uniformity, conserving nutrient soil characteristics, and increasing productivity per unit of area [28,65]. In contrast, continuous grazing promotes greater herbage selection and intake if animals have a similar herbage allowance [31]. In our review, the grazing method did not show consistent results on CH 4 emissions ( Table 1). In one experiment, increased CH 4 intensity was associated with decreased animal production in rotational vs. continuous stocking, related to lower intake [31]. Under wellmanaged grazing conditions, no differences were observed in continuous or rotational grazing in herbage accumulation, forage nutritive value, intake, or performance because the animal can select and consume forages of greater nutritional value [64]. The limited number of studies comparing continuous and rotational grazing limits the evaluation of the effects of rotational or continuous methods on CH 4 emissions. Additionally, to our knowledge, no studies have evaluated other grazing methods such as first-last grazers [64] on CH 4 emissions.
Another strategy to increase herbage mass is applying nitrogen fertilization [54,66], allowing a greater stocking rate and extending the grazing period [59]. Nitrogen fertilization did not modify enteric CH 4 production except with nitrogen application rates greater than 400 kg/ha [45]. However, nitrogen application did not modify forage biomass or nutritive value, explaining the absence of effects on CH 4 yield or intensity (Table 1). No experiments have evaluated the effects of fertilization with other nutrients on CH 4 production.
Legume inclusion in pastures has important advantages for grassland systems. Legumes can fix atmospheric nitrogen through the symbiotic relationship with Rhizobium bacteria and increase crude protein concentration in forage diets [67]. Legumes are C3 species and have more digestible tissue (i.e., mesophyll), greater crude protein concentration, and greater microbial degradation than C4 grasses [62,68]. Several studies have been conducted to evaluate the effects of legumes on CH 4 production. There are differences in ruminant CH 4 emissions when the legume evaluated contains condensed tannins. Generally, nontanninferous legumes such as Medicago sativa or Trifolium spp. Have a greater extent of ruminal organic matter digestion, especially in low-quality grass-based diets [50]. Their inclusion in ruminants' diets increase dry matter intake and animal performance [48,52,53,69]. However, in most retrieved manuscripts, non-tanninferous legumes did not modify CH 4 production, yield, or intensity (Table 1). Factors such as legume proportion in pasture, dry matter intake, organic matter digestibility, and passage rate can help explain the absence of any effects on CH 4 emissions. For example, McCaughey et al. [52] reported that 78% of alfalfa in the pasture increased dry matter intake by 13% and decreased CH 4 production by 10%. In contrast, Chaves et al. [49] reported that 40% of alfalfa in the pasture did not affect intake or CH 4 production.
Tannin-containing legumes such as Lotus spp. or Calliandra calothyrsus contain polyphenol compounds that protect plants against external stressors. Variations in tannin type, concentration, and activity depend on environmental conditions and management practices [70]. Waghorn [71] suggested that tannins reduce ruminal protein and carbohydrate fermentation and microbial enzyme activity and affect methanogenic archaea populations [70,72]. Thus, CH 4 emissions may be reduced due to decreased nutrient fermentation, diminished dihydrogen (H 2 ) production, or a modified archaeal community. However, high tannin concentration is associated with detrimental effects on animal performance, related to decreased dry matter intake and protein digestibility [71]. There is too little information to conclude on the effect of the inclusion of tannin-containing legumes as a strategy to modify CH 4 emissions ( Table 1).
Silvopastoral systems are a spatial arrangement where multiple forage strata grow together to provide forage biomass and other ecosystem services [73]. There are multiple silvopastoral designs where woody species may or may not provide forage for ruminant diets [74]. Silvopastoral systems decrease the nutritive value of grass and biomass production because trees can intercept light, increase cell wall concentration, and reduce herbage photosynthetic ability [75]. However, in warm or dry conditions, the presence of trees produces a micro-environment that maintains forage production, reduces maturity, and increases nutritive value [73]. There are few evaluations of silvopastoral systems on enteric CH 4 emissions. Ruminants in silvopastoral systems produced similar CH 4 emissions to ruminants in grasslands without trees due to similar forage nutritive value and animal intake ( Table 1). The foliage of some shrubs and trees has secondary compounds, such as tannins or saponins, that have been reported to decrease CH 4 emissions from ruminants; however, this has not been evaluated under grazing conditions [76].

Supplementation Strategies to Mitigate Methane Emissions from Ruminants in Grasslands Systems
Diet supplementation is a nutritional strategy to supply deficient nutrients, improve the health status, increase animal performance, and reduce GHG emission intensity, especially in undernourished ruminants [77]. Concentrate supplementation at pasture showed contradictory results on CH 4 emissions ( Table 2). It is expected that concentrate supplementation increases rumen fermentation of forage diets, resulting in greater absolute production of H 2 and CH 4 in the rumen [78]. Thus, concentrate supplementation may increase the total CH 4 production (i.e., total grams of CH 4 per animal per day). Conversely, concentrate supplementation increases ruminal passage and reduces the rumen pH, resulting in lower CH 4 relative to organic matter fermented because the hydrogen is redirected to other metabolic pathways (i.e., propionate or microbial bacteria synthesis; [79,80]. In addition, methanogens are sensitive to ruminal pH lower than 6 [81]. Thus, concentrate supplementation might decrease CH4 yield (i.e., total grams of CH 4 per unit of OM fermented). Finally, concentrate supplementation increases dry matter intake and animal performance resulting in lower CH 4 intensity (i.e., total grams of CH 4 per unit of product), and effects appear to be dependent on the level of concentrate supplied. Lipid supplementation is another strategy to increase the energetic density of ruminant diets, especially during high-energy demand periods such as early lactation [97], which has shown promise as a CH 4 mitigation strategy [98]. Lipid supplementation under grazing conditions decreased CH 4 intensity 60% (Table 2). Lipid inclusion may reduce fiber digestion, by coating the fiber against microbial fermentation [14,99]. Fiber fermentation is related to acetate and H 2 production in the rumen. In this regard, reducing dry matter intake and fiber degradability potentially decreases H 2 and CH 4 production, though it is not a desirable mechanism to reduce CH 4 emissions [78]. Furthermore, fatty acids can inhibit methanogens. Poly-unsaturated and medium chain fatty acids have toxic and disruptive effects on methanogen cell membranes [98,100]. In addition, unsaturated fatty acids capture H 2 during the rumen biohydrogenation process, although this represents a small proportion of H 2 capture [12]. Finally, nitrates are reduced in the rumen to ammonia, competing with methanogenesis [9]. However, nitrates did not reduce CH 4 emissions of ruminants under grazing conditions (Table 2).

Perspectives on Methane Mitigation Strategies in Grasslands Systems
Designing effective mitigation strategies of CH 4 emissions under grazing conditions represents a significant challenge, especially in extensive systems. These challenges are reflected in the fact that considerably less research has been conducted under grazing conditions, in which there is less control of the experimental conditions compared to confined systems. In addition, the heterogeneity and seasonal variation in grazing lands' forage composition and growth further complicate conditions in these systems by adding experimental variation. The most widespread technique for measuring CH 4 under grazing conditions is the SF 6 tracer technique, which is less precise and more labor intensive than respiration chambers [17,18]. The inability to accurately determine intake in grazing conditions hinders the ability of researchers in terms of assessing CH 4 emissions yield and the impact of interventions on forage intake. Thus, results of CH 4 abatement strategies evaluated in grazing systems are less consistent than in confined studies, and researchers may need to rely on the impact on emissions intensity rather than yield when assessing these strategies. Animal performance (i.e., milk yield or growth) is frequently and more precisely evaluated during grazing experiments. Therefore, CH 4 intensity should be a more useful variable to compare practices in grazing conditions. This review highlights CH 4 mitigation practices that have been studied under grazing conditions. Emission of CH 4 may be reduced through grazing practices that modify the forage composition (i.e., reducing structural carbohydrate intakes) as a result of an increased stocking rate or lower pre-grazing herbage mass. Rotational grazing does not increase emissions intensity unless animal intake is restricted, compromising production. Decreasing forage maturity and the presence of tannin-containing legumes can decrease CH 4 emissions; however, more research is required under grazing conditions to further quantify this. Non-tanniferous legumes mostly did not modify CH 4 emissions. Additionally, nitrogen fertilization and silvopastoral systems have had no effects on CH 4 emissions. Conversely, concentrate and lipid supplementation of grazing diets have improved animal performance and reduced CH 4 intensity. Nitrates supplementation has not shown a consistent effect on CH 4 production from grazing ruminants. Supplementation can be problematic in extensive systems where infrequent animal management and low profitability restricts its implementation [11,25]. Other additives such as tannins, 3-nitrooxypropanol, or red algae have shown promising results on CH 4 reduction under confined conditions [101,102], but their effects on grazing systems are as yet uncertain. Finally, long-term studies and integrative evaluation through life cycle assessment analysis are needed to generate technologies that promote greater biological efficiency and farm profitability while reducing the detrimental effects on the environment.

Data Availability Statement:
The data presented in this paper are available on request from corresponding author.